重组犬IFN-γ的原核表达及生物学活性研究

Study on prokaryotic expression and biological activity of recombinant canine IFN-γ

为了探究犬γ干扰素(canine interferon gamma,CaIFN-γ)的原核可溶性表达方法及其生物学活性,试验首先采用PCR方法扩增优化后的CaIFN-γ基因成熟肽序列,以此构建重组表达质粒pCold-SUMO-10His-CaIFN-γ;然后将重组表达质粒转化至表达菌株Lyophilized BL21(DE3)Chaprone中,并用IPTG进行诱导表达;最后对表达的重组蛋白rCaIFN-γ进行SDS-PAGE分析、Western-blot鉴定、IPTG浓度优化、Ni-IDA亲和层析,以及采用CCK-8法测定重组蛋白rCaIFN-γ的细胞毒性,实时荧光定量PCR(相对定量)法检测凋亡相关基因和干扰素刺激基因(ISGs)转录水平变化,用细胞病变(CPE)法测定抗水疱性口炎病毒(VSV)效价,实时荧光定量PCR(绝对定量)法测定抗犬流感病毒(CIV)H3N2亚型感染的能力。结果表明:优化后的CaIFN-γ基因PCR扩增产物大小为494 bp,且重组表达质粒pCold-SUMO-10His-CaIFN-γ经菌液PCR、双酶切和测序验证构建成功;阳性菌株经诱导后表达的重组蛋白rCaIFN-γ大小为35.1 ku,该蛋白质能以可溶性形式表达,且可以与His Tag和HA Tag的鼠源单克隆抗体发生特异性反应;IPTG最佳诱导表达浓度为0.05 mmol/L;纯化后的重组蛋白rCaIFN-γ最大安全浓度为20μg/mL。以最大安全浓度的重组蛋白rCaIFN-γ分别作用MDCK细胞、MDCK(PB_(2))细胞4 h和6 h后,促凋亡基因p53和Bcl2相关X蛋白(Bax)的相对表达量无显著变化(P>0.05),γ干扰素受体2(IFNGR2)基因相对表达量降低(P<0.01或P>0.05),信号转导及转录激活因子1(STAT1)和干扰素调节因子1(IRF1)基因相对表达量均升高(P<0.05或P<0.01);MDCK细胞中,作用6 h IFNGR2、STAT1和IRF1基因相对表达量略高于4 h,但差异不显著(P>0.05);MDCK(PB_(2))细胞中,作用6 h IFNGR2和IRF1基因相对表达量极显著高于4 h(P<0.01),而STAT1基因相对表达量显著低于4 h(P<0.05)。再以最大安全浓度的重组蛋白rCaIFN-γ分别刺激MDCK和MDCK(PB_(2))细胞24 h,干扰素诱导跨膜蛋白1(IFITM1)、抗黏病毒蛋白1(MX1)、干扰素诱导蛋白与四肽重复1(IFIT1)、干扰素刺激基因15(ISG15)、2′-5′寡腺苷酸合成酶1(OAS1)5种ISGs转录水平均升高,且MDCK细胞中的转录水平均极显著高于MDCK(PB_(2))细胞(P<0.01)。该蛋白质在MDCK细胞中抗VSV的病毒活性效价为6.4×10~5 U/mL,并且对H3N2亚型CIV有一定的抑制作用。说明重组蛋白rCaIFN-γ在大肠杆菌中成功地实现了可溶性表达,并且具有良好的生物学活性。

In order to investigate the prokaryotic soluble expression method and biological activity of canine interferon gamma(CaIFN-γ),in this experiment,the optimized mature peptide sequence of CaIFN-γgene was amplified by PCR to construct the recombinant expression plasmid pCold-SUMO-10His-CaIFN-γ.The recombinant expression plasmid was transformed into Lyophilized BL21(DE3)Chaprone and induced by IPTG.Finally,SDS-PAGE analysis,Western-blot identification,IPTG concentration optimization,Ni-IDA purification and CCK-8 method were used to determine the cytotoxicity of recombinant rCaIFN-γ.Real-time fluorescent quantitative PCR(relative quantitative)was used to detect the transcriptional changes of apoptosis-related genes and interferon-stimulated genes(ISGs),the titer of anti-vesicular stomatitis virus(VSV)was determined by microcytopathogenic inhibition(CPE),and the ability of anti-Canine influenza virus(CIV)H3N2 subtype infection was determined by real-time fluorescent quantitative PCR(absolute quantitative).The results showed that the optimized PCR amplification product of CaIFN-γgene was 494 bp in size,and the recombinant expression plasmid pCold-SUMO-10His-CaIFN-γwas successfully constructed by bacterial solution PCR,double enzyme digestion and sequencing.The recombinant protein rCaIFN-γexpressed by the positive strain after induction was 35.1 ku in size,which could be expressed in a soluble form and could specifically react with His Tag and HA Tag labeled murine monoclonal antibodies.The optimal induced expression concentration of IPTG was 0.05 mmol/L.The maximum no-cytotoxic concentration of the purified recombinant protein rCaIFN-γwas 20μg/mL.After treating MDCK cells and MDCK(PB_(2))cells with the maximum safe concentration of recombinant protein rCaIFN-γfor 4 h and 6 h,the relative expressions of pro-apoptotic genes p53 and Bax had no significant changes(P>0.05);the relative expressions of IFNGR2 genes were decreased(P<0.01 or P>0.05),and the relative expressions of STAT1 and IRF1 genes were increased(P<0.05 or P<0.01).The relative expression levels of IFNGR2,STAT1 and IRF1 genes in MDCK cells at 6 h were slightly higher than those at 4 h,but the differences were not significant(P>0.05).In MDCK(PB_(2))cells,the relative expression of IFNGR2 and IRF1 genes at 6 h was significantly higher than that at 4 h(P<0.01),while the relative expression of STAT1 gene at 6 h was significantly lower than that at 4 h(P<0.05).Then,MDCK and MDCK(PB_(2))cells were stimulated with the maximum safe concentration of recombinant protein rCaIFN-γfor 24 h,respectively,and the transcription levels of IFITM1,MX1,IFIT1,ISG15 and OAS1 increased.The transcription levels of MDCK cells were significantly higher than those of MDCK(PB_(2))cells(P<0.01).The titer of the protein against VSV in MDCK cells was 6.4×10^(5) U/mL,and it had certain inhibitory effect on H3N2 subtype CIV.These results indicated that the recombinant protein rCaIFN-γwas successfully expressed in E.coli and had good biological activity.